Process for producing and multiplying lymphocytes

ABSTRACT

A process is disclosed for growing and/or multiplying lymphocytes in a cell culture medium which contains a lymphocyte growth factor, as well as aurintricarboxylic acid, cyclosporin and/or ascomycin. This process is particularly suitable to produce tumoricide killer T-cells.

[0001] The invention concerns a process for producing and multiplyinglymphocytes as well as a composition which is suitable as a culturemedium for lymphocytes.

[0002] The production and multiplication of lymphocytes is problematic.Some types of lymphocytes cannot be cultured at all in vitro or are verydifficult to culture in vitro. Native B lymphocytes can for example onlybe cultured for a short period and T lymphocytes require difficultculture conditions for longer culture such as a combination of growthfactor(s) and “feeder cells” (“nurse cells”) or the use ofunphysiological and potentially dangerous substances such as tumourpromoters (phorbol esters) in combination with ionophoric substances(e.g. ionomycin) or plant lectins (e.g. phytohaemagglutin, PHA). Thisputs severe constraints on possibilities for the long-term culture andmultiplication of T lymphocytes and other lymphocytes, and theproduction of lymphocytes in significant amounts for diagnostic ortherapeutic purposes is very limited or impossible.

[0003] However, there is manifold interest in the ability to produce,culture and multiply lymphocytes in vitro e.g. B lymphocytes asproducers of specific antibodies or cytotoxic T lymphocytes (CTL) totreat infections or tumours, in addition regulator T lymphocytes (helperor suppressor T lymphocytes) for the diagnosis and treatment ofautoimmune diseases or “natural killer (NK) lymphocytes for treatingmalignant growths.

[0004] Hybridoma cells which are formed from a fusion of B lymphocytesor T lymphocytes with malignant, lympoid cells (e.g. myeloma cells) canbe cultured and multiplied without difficulty. Such hybridoma cells havethe immunological function of the original lymphocytes as well as theessentially unlimited ability to proliferate of the malignant fusionpartner. However, the application of the hybridoma technique is limitedto a few animal species (mouse, rat) and essentially fails in othermammalian species and also in particular in humans.

[0005] A process for the production of proliferating CD4+ lymphocytes isdescribed in WO90/10059. According to this peripheral mononuclear bloodcells (PBNMC) are treated with alkyl esters to remove monocytes andgranulocytes and subsequently cultured in a culture medium whichcontains a T cell stimulant and/or IL-2. Mitogens such as PHA are usedas the T cell stimulant. However, the use of IL-2 alone only leads to alow proliferation of the cells. The addition of mitogenic substancesduring the culture of cells which are subsequently to be implanted intoa patient is critical.

[0006] A process for the culture of T cells in the presence ofinterleukin-2 is also described in EP-A 0 203 403. A disadvantage ofthis process is also that the T cells can only be proliferated to aslight extent by this means.

[0007] A process for culturing and multiplying tumoricidal T lymphocytesis described in WO94/23014 by co-culturing lymphocytes with a cell line(stimulator cells) while avoiding an allogenic stimulation and withoutaddition of interleukin-2. In this process resting T lymphocytes areactivated to effector cells which recognize and kill tumour cells orinhibit their growth. A considerable amount of fermentation is requiredto provide such stimulator cells for the mass proliferation of forexample tumoricidal killer T cells. In addition the killer T cells mustbe separated in a sterile manner from these stimulator cells or theircell debris before use (reinfusion into the patient).

[0008] V. Kutnik et al., Period. Biol. 92 (1990) 48 describe that, inthe allogenic mixed lymphocyte reaction of mouse spleen lymphocytes, theaddition of IL2 restores the cyclosporin A-induced inhibition of theproliferation of the responder cells and increases their alloreactivity.

[0009] Pierson, B. A. et al., Blood 87 (1996) 180-189 describe thatisolated human NK cells (CD56+, CD3−) can be multiplied to a slightextent in culture by adding IL2. The addition of supernatants ofirradiated mononuclear cells from blood increases the multiplication ofthe NK cells. Thrombospondin was identified as the active principle ofthis effect which does not act directly but rather indirectly byactivation of latent TGFβ. TGFβ activated in this manner inhibits theproliferation in the early phase of the culture and increases thefurther proliferation. A repeated addition of TGFβ when the medium ischanged inhibits the growth of the cells and in this case suppresses theproliferation of the NK cells.

[0010] In Immunological Invest. 25 (1996) 129-151 I. A. Ayoubinvestigates the effect of human TGFβ on a bovine CD4+ lymphoblastoid Tcell line (BLTC) which grows autonomously in IL2-containing medium. Thecell line is arrested in medium without IL2. The addition of TGFβ to thearrested BLTC drives them rapidly into apoptosis. The simultaneousaddition of IL2 abolishes the arrest and prevents the induction ofapoptosis. The addition of TGFβ to suboptimal concentrations of IL2co-stimulates the proliferation of the BLTC.

[0011] In Intern. Immunol. 6 (1994) 631-638 R. de Jong describes theeffect of TGFβ1 on the proliferation of isolated subpopulations of humanCD4+ T lymphocytes. TGFβ1 amplifies the proliferation of CD4 cells (CD45RA+) by antibodies in the presence of IL2, but the proliferation ofpre-activated T cells (CD45 RO+) is inhibited by the addition of TGFβ1.However, it turned out that the induced proliferation of the CD45RA+cells is inhibited after five days by addition of TGFβ1.

[0012] A. Cerwenka describes in J. Immunol. 156 (1996) 459-464 that thepresence of TGFβ1 during the primary stimulation of human T lymphocytesincreases their ability to survive in secondary cultures and reducestheir susceptibility to apoptosis-inducing anti-Fas antibodies. Theaddition of TGFβ1 also reduces the apoptosis susceptibility of primaryactivated T lymphocytes to secondary activation. A survival of the Tcells over a long period is ensured in the presence of IL2 and TGFβ1.

[0013] T. H. Inne et al., J. Immunol. 148 (1992) 3847-3856 describe thatthe proliferation of CTLL-2 cells (murine T cell line) in IL2-containingmedium is inhibited by addition of TGFβ1. In addition to the inhibitionof proliferation, TGFβ1 induces a change in the cell morphology andinduces the expression of the surface molecule CD8 in CTLL-2. Acombination of IL2 and TGFβ also induces an increased expression of CD8in murine thymocytes which have been activated by phorbol dibutyrate andionomycin. In this case the addition of TGFβ1 also reduces theproliferation rate.

[0014] However, TGFβ is not a substance that is readily and cheaplyavailable in adequate amounts. TGFβ is usually either isolated fromnatural sources or produced recombinantly. The main object of theinvention was to provide an effective and cheap means for culturing andmultiplying lymphocytes as well as a process for the production ofpancytotoxic T cells. A further object of the present invention is toprovide a process which enables lymphocytes to be produced, culturedand/or multiplied in a simple manner on a large scale.

[0015] The subject matter of the invention is a process for culturingand/or multiplying lymphocytes in a cell culture medium which contains alymphocyte growth factor and additionally aurintricarboxylic acid,cyclosporin and/or ascomycin.

[0016] Surprisingly the process according to the invention enableslymphocytes with special properties to be produced in a simple manner,to be cultured over a long period and to be multiplied on a considerablescale. The process according to the invention is particularly suitablefor culturing and multiplying T lymphocytes and NK lymphocytes.

[0017] Furthermore it has turned out that the process according to theinvention particularly advantageously enables lymphocytes to be culturedover a long period (more than 14 days) and to be multiplied on a largescale (factor of 100, 1000 or more). In addition a combination of alymphocyte growth factor and additionally aurintricarboxylic acid,cyclosporin and/or ascomycin enables pancytotoxic T cells to bemultiplied from lymphocyte mixtures by long-term culture, preferablyfrom a lymphocyte cell population and hence enables such cells to beproduced simply and in large amounts.

[0018] A lymphocyte growth factor is understood as a substance which isable to promote cell division of lymphocytes. Lymphocyte growth factorsare known to a large extent to a person skilled in the art. Suitable Tlymphocyte growth factors are for example interleukin 1 (IL-2) andinterleukin 15 (IL-15). A suitable NK lymphocyte growth factor is e.g.IL-15. Suitable B lymphocyte growth factors are for example interleukin13 (IL-13), IL-14 and IL-10 (Callard, R. E., and Gearing, J. H., TheCytokine Facts Book, Academic Press, London, 1994).

[0019] Surprisingly Cyclosporin® (e.g Cyclosporin A®), Ascomycin®(FK520) and/or Tacrolimus®(FK506) can be used according to the inventionto increase proliferation. These are substances which bind to acyclophilin and inhibit calcineurin in this complex. Other substanceswhich have these properties are also suitable according to theinvention. Pazderka-F., et al., Transpl. Immunol. (1996) 23-31,Rusnak-F., et al., Bone-Marrow-Transplant 17 (1996) 309-13, Su-Q., etal., Ren-Physiol-Biochem. 18 (1995) 128-39, Baughman-G., et al.,Mol-Cell-Biol. 15 (1995) 4395-402, Kawamura, A., et al., J-Biol-Chem.270 (1995) 15463-6, Kakalis, L T., et al., FEBS-Lett. 362 (1995) 55-8.Surprisingly aurintricarboxylic acid (ATA) can also be used to increasethe proliferation of lymphocytes. Aurintricarboxylic acid is a substancewhich inhibits the interaction of proteins and nucleic acids [Gonzales,R. G. et al.: Biochemistry 19: 4299-4303 (1980)] and is a generalinhibitor of nucleases.

[0020] The amount of aurintricarboxylic acid which is added in theprocess according to the invention can also be varied and is to acertain extent dependent on the medium used and on the cell to becultured. It has turned out that for example when culturingtumour-infiltrating lymphocytes (TIL) in serum-containing medium it isadvantageous to add 0.1-100 μM ATA.

[0021] Lymphocytes within the sense of the invention are understood asleucocytes which are derived from lymphocyte progenitor cells in thehaematopoietic system and can be for example found in blood, in thelymph, in the spleen, in lymph nodes, in tumours (tumour-infiltratinglymphocytes, TIL) or inflamed tissue. Important subgroups are Tlymphocytes, NK lymphocytes and B lymphocytes. B lymphocytes areantibody-producing lymphocytes in their mature form.

[0022] T lymphocytes (T cells) are understood as lymphocytes which arefor example involved in cell-mediated cytotoxicity, in allergy of thedelayed type and in the activation of B lymphocytes. There are numerousdifferent types (subtypes) of T cells which can be each distinguished bytheir function and/or their cell surface antigens (see e.g. Imm. Rev.(1993), 74 and (1992), 82; Advances in Immunology 58 (1995) 87). Suchsurface antigens are for example referred to as CD (Cluster ofDifferentiation) antigens. The expression of the antigen-recognizing Tcell receptor is typical for all T cells. T cells develop fromhaematopoietic stem cells and mature, with some exceptions, in thethymus. Examples of T cells are cytotoxic T cells, helper T cells,suppressor T cells, suppressor-inducer T cells and killer T cells.

[0023] Natural killer cells (NK cells) are lymphoid cells which developfrom haematopoietic stem cells and differ from T cells and B cells inthat they express neither the T cell receptor nor the B cell receptorand are CD3−.

[0024] The culture of lymphocytes in the process according to theinvention is carried out in a conventional basic culture medium whichadditionally contains a lymphocyte growth factor and cyclosporin and/orascomycin. All media are suitable as basic culture media which areusually used to culture mammalian cells. Such culture media can eithercontain serum or be serum-free and are known to any person skilled inthe art. Examples are RPMI 1640-medium, Dulbecco's Modified EaglesMedium (DMEM), F12 medium or a mixture of the latter (DF medium) whichcan be used in a serum-containing and also in a serum-free form. Ifserum-free media are used, the medium must be supplemented by criticalcomponents. Such critical components are, as known to any person skilledin the art, albumin, transferrin, selenite and insulin. Serum-free mediawhich already contain all critical supplements such as e.g. the culturemedium X-Vivo 20® (Bio-Whittaker, Serva) are also particularly suitable.The amounts of lymphocyte growth factor and cyclosporin and/or ascomycinwhich are added in the process according to the invention can vary and,to a certain extent, depend on the medium used (serum-free orserum-containing) and on the cell to be cultured. It has turned out thatin serum-free culture amounts of 0.1-10×10⁻⁹ mol/l growth factor and10⁻¹⁰-10⁻² mol/l cyclosporin and/or ascomycin are suitable. Consequentlyin the culture of T cells and/or NK cells in serum-free medium it isadvantageous to for example add 10-20 ng/ml IL-2 or IL-15 and 5-20 ng/mlCyclosporin-A® and/or 1-10 ng/ml Ascomycin®.

[0025] The process according to the invention is especially suitable forculturing and multiplying killer T cells (KT cells) and tumoricidalkiller T cells. Such tumoricidal killer T cells can for example beproduced according to WO 94/23014 by co-culturing lymphocytes, which forexample have been isolated from blood, with stimulator cells. In thisprocess resting T lymphocytes are activated to effector cells whichrecognize and kill tumour cells and inhibit their growth. The activatedT lymphocytes are stimulated to proliferate in this co-culture and canbe further cultured and multiplied by the process according to theinvention.

[0026] The process according to the invention is also advantageous forthe production and multiplication of pancytotoxic T cells. A combinationof a lymphocyte growth factor with aurintricarboxylic acid, and asubstance which binds to cyclophilin and inhibits calcineurin in thiscomplex, or an apoptosis antagonist is suitable. In addition the processaccording to the invention enables the sustained proliferation of bloodlymphocytes after treatment with leucyl-leucine-methyl ester. Thephenotyping of such cells produced according to the invention on thebasis of surface markers shows that the proliferating cells areuniformly T lymphocytes (100% CD3+). These cells exhibit a previouslyunknown activity which is referred to as pancytotoxic activity in thefollowing. Pancytotoxic T cells are characterized in that theyindiscriminately kill normal cells such as e.g. fibroblasts,keratinocytes or endothelial cells and also tumour cells such as e.g.malignant melanoma, T lymphoma or lung carcinoma. Surprisinglypancytotoxic T cells can be produced from mononuclear cells by treatmentwith a combination IL-2 and apoptosis antagonists. Pancytotoxic T cellscan be used advantageously for the local treatment of tumours (e.g.tumour metastases).

[0027] An apoptosis antagonist (apoptosis inhibitor) is to be understoodas a substance which is able to not allow the genetically determinedself-destruction program to become effective in a cell that would leadto cell death after activation, and which partially or completely slowsor prevents the lysis of a cell after activation of a suicide signal.Suitable substances are for example described in Kroemer, G., Advancesin Immunology 58 (1995) 211-296. According to the invention substancesare suitable which are able to inhibit agents with an apoptosis signaleffect on lymphocytes (e.g. an antibody to TNFα which prevents bindingof this cytokine to its receptor). Furthermore substances are suitablewhich prevent reception of an apoptosis signal by the lymphocytes (e.gantibody to the TNFα receptor which inhibits binding of TNFα to thisreceptor). Substances are also suitable which are able to preventapoptosis by interrupting the signal chain from the cell membrane intothe inside of a lymphocyte (e.g. an inhibitor of sphingomyelinase,inhibition of the formation of ceramide). Finally an apoptosis inhibitoris also to be understood as a substance which is able to activate theanti-apoptosis program in a lymphocyte which is also geneticallydetermined (e.g. up-regulation of the bcl-2 expression). Cyclosporin andascomycin are also suitable as calcineurin inhibitors.

[0028] It has surprisingly turned out that when mononuclear blood cells(PBMNC) are cultured without pre-treatment with leucyl-leucine-methylester, lymphocytes usually grow after a latency period of 14-28 dayswhich can be multiplied as desired. Phenotyping of these cells showsthat they represent a mixture of T cells (CD2+, CD3+) and NK cells(CD2+, CD3−, CD16+). Functionally they are also pancytotoxic and thusdiffer from KT cells. In this case it is also preferable to use IL2 orIL15 as a lymphocyte growth factor and Cyclosporin or Ascomycin.

[0029] The following examples, publications and the figures furtherelucidate the invention, the scope of which results from the patentclaims. The described processes are to be understood as examples butstill describe the subject matter of the invention even aftermodifications.

[0030]FIG. 1 shows the growth behaviour of killer T cells in serum-freeDF medium on addition of IL-2+ CsA(A) and CsA alone (B).

[0031]FIG. 2 shows the growth behaviour of killer T cells in serum-freeDF medium on addition of IL-15 and CsA(A) and IL-15 alone (B).

[0032]FIG. 3 shows the growth behaviour of killer T cells in serum-freeDF medium on addition of IL-2 and Ascomycin (FK520) (A) and Ascomycinalone (B).

[0033]FIG. 4 shows the growth behaviour of killer T cells in serum-freeDF medium on addition of IL-2 and aurintricarboxylic acid (A) andarintricarboxylic acid alone (B).

EXAMPLE I

[0034] Production of Killer T Cells

[0035] Mononuclear cells from peripheral blood (PBMNC) of human donorsare isolated by means of gradient centrifugation (lymphocyte separationmedium, BM), washed twice with phosphate-buffered saline solution andincubated at a density of 5-10×10⁶ cells/ml DF medium according toThiele and Lipsky (J. Immunol. 136 (1986) 1038-1048) with 250 μMleucyl-leucine-methyl ester (BM) for 20 minutes at room temperature.After washing with DF medium the cells are cultured at 37° C. in 8% CO₂atmosphere at a density of 1-2×10⁶ per ml DF medium together withirradiated (2000 rad) HB654 cells or HB617 cells (stimulator cells;2-5×10⁵ per ml). On day 5-6 of the co-culture half to two thirds of theculture medium is renewed and irradiated stimulator cells (2-5×10⁵ perml) are added again. From day 8-10 after starting the co-culture, whenall stimulator cells have been destroyed by the cytotoxic activity ofthe killer T cells, the killer T cells are used for the followingexamples.

[0036] Phenotyping the cells on day 10 of the co-culture shows that >95%of the cells are CD3+, ca. 40% CD4+ and ca. 60% CD8+. Cells with themarkers CD19 or CD16 are not found.

EXAMPLE 2

[0037] Multiplication of Killer T Cells in Serum-free Medium whichContains Interleukin-2 (IL-2) and Cyclosporin A (CsA).

[0038] Killer T cells which have been produced according to example 1are washed once in DF medium and cultured at a density of 5×10⁵ per mlDF medium in two separate preparations that are denoted A and B. Humanrecombinant IL-2 (BM; 20 ng/ml) and CsA (Sandoz; 12.5 ng/ml) are addedto preparation A. Only CsA (12.5 ng/ml) is added to preparation B. Halfof the culture medium is renewed every second day and the cell count isadjusted to 5×10⁵ per ml.

[0039] As shown in FIG. 1 the killer T cells multiply in preparation A(IL-2+CsA) with a doubling time of about 48 hours for at least tendoubling cycles (corresponds to 1000-fold multiplication). Inpreparation B (only CsA) the killer T cells do not multiply.

EXAMPLE 3

[0040] Multiplication of Killer T Cells in Serum-free Medium whichContains Interleukin-15 (IL-15) and Cyclosporin A (CsA).

[0041] Killer T cells which have been produced according to example 1are washed once in DF medium and cultured at a density of 5×10⁵ per mlDF medium in two separate preparations that are denoted A and B. Humanrecombinant IL-15 (R & D Systems; 15 ng/ml) and CsA (Sandoz; 12.5 ng/ml)are added to preparation A. Only IL-15 (15 ng/ml) is added topreparation B. Half of the culture medium is renewed every second dayand the cell count is adjusted to 5×10⁵ per ml.

[0042] As shown in FIG. 2 the killer T cells multiply in preparation A(IL-15+CsA) with a doubling time of about 48 hours for at least 11doubling cycles. In preparation B (only IL-15) the proliferationstagnates after 3 doubling cycles.

EXAMPLE 4

[0043] Multiplication of Killer T Cells in Serum-free Medium ContainingInterleukin-2 (IL-2) and Ascomycin (FK 520).

[0044] Killer T cells which have been produced according to example 1are washed once in DF medium and cultured at a density of 5×10⁵ per mlDF medium in two separate preparations that are denoted A and B. rh IL-2(20 ng/ml) and Ascomycin (Calbiochem; 2.5 ng/ml) are added topreparation A. Only Ascomycin (2.5 ng/ml) is added to preparation B.Half of the culture medium is renewed every second day and the cellcount is adjusted to 5×10⁵ per ml.

[0045] As shown in FIG. 3 the killer T cells multiply in preparation A(IL-2+Ascomycin) with a doubling time of about 48 hours for at least tendoubling cycles. In preparation B (only Ascomycin) no multiplication ofkiller T cells is observed.

EXAMPLE 5

[0046] Multiplication of NK and T Lymphocytes from Mononuclear Cells ofHuman Blood in Serum-free Medium which Contains Interleukin-2 (IL-2) andTransforming Growth Factor-β1 (TGF-β1).

[0047] Mononuclear cells from peripheral blood (PBMNC) of a human donorare isolated by means of gradient centrifugation (lymphocyte separationmedium, Boehringer Mannheim GmbH, Germany (BM)), washed twice withphosphate-buffered saline solution and taken up in DF medium at adensity of 1×10⁶ cells/ml and cultured in three separate preparationswhich are denoted A, B and C. Recombinant human IL-2 (BM, 20 ng/ml) andrh TGFβ1 (BM, 4 ng/ml) are added to preparation A, only IL-2 (20 ng/ml)is added to preparation B and only TGFβ1 (4 ng/ml) is added topreparation C. Half of the culture medium (+cytokineis)) is renewedevery second day and the cell count is adjusted to ca. 1×10⁶ cells/mlduring the first 7 days and subsequently to ca. 0.5×10⁵ cells/ml.

[0048] In preparation A (IL-2+TGFβ1) the number of non-adherent lymphoidcells increases approximately from day 5 after the beginning of theculture, firstly with a doubling time of ca. 96 hours, from day 20 whenno more colonies with adherent monocytic cells are detectable with adoubling time of less than 48 hours. On day 50 of the continuous culturethe cell count has increased more than 10⁴-fold compared to the initialstate. The analysis of the cells with regard to their surface markersyields the following result on day 50: the population contains:

[0049] ca. 50% NK cells (CD2+, CD3−, CD16+, CD56+) and

[0050] ca. 50% T cells (CD3+, CD4+, CD8+)

[0051] In preparation B (only IL-2) lymphoid cells only multiplymoderately (<50-fold) over ca. 16 days, then stagnate and die after ca.20 further days.

[0052] In preparation B (only TGFβ1) the cells do not multiply.

EXAMPLE 6

[0053] Multiplication of Killer T Cells in Serum-free Medium ContainingInterleukin 2 (IL-2) and Aurintricarboxylic Acid (ATA).

[0054] Killer T cells which have been produced according to example 1are washed once in DF medium and cultured at a density of 5×10⁵ per mlDF medium in two separate preparations that are denoted A and B. IL-2(20 ng/ml) and aurintricarboxylic acid (Aldrich Chemie; 4.2 μg/ml) areadded to preparation A. Only aurintricarboxylic acid (4.2 μg/ml) isadded to preparation B. Half of the culture medium is renewed everythird day and the cell count is adjusted to 5×10⁵ per ml.

[0055] As shown in FIG. 4 the killer T cells multiply in preparation A(IL-2+ATA) with a doubling time of about 72 hours for at least 11doubling cycles. In preparation B (only ATA) the killer T cells do notmultiply.

EXAMPLE 7

[0056] Multiplication of Tumour-infiltrating Lymphocytes in a Mediumwhich Contains Interleukin-2 (IL-2) and Aurintricarboxylic Acid (ATA).

[0057] The nodules of a human colon carcinoma which was removed by anoperation from the large intestine is freed of connective tissue andnormal parts of the intestine and cut into ca. 2×2×3 mm pieces. Thetumour fragments are taken up in Iscove-modified DME medium (Gibco)which contains 15% FCS (BM) and divided equally into four culture disheswhich are named A, B, C and D. IL-2 (20 ng/ml) is added to preparationA, aurintricarboxylic acid (4.2 gg/ml) is added to preparation B andIL-2 (20 ng/ml) plus aurintricarboxylic acid (4.2 μg/ml) is added topreparation C. Preparation D remains without additions. Three quartersof the respective culture media is removed each second day withoutchanging the tissue or cell content. Microscopic control of the culturesshows that after 24-48 hours lymphocytic cells migrated out of thetumour tissue fragments which multiplied in the partial culture C whichcontains IL-2 plus ATA but not in the partial cultures A, B or D. Aftera ten day culture the picture is as follows: In A and B (only IL-2 orATA) the number of emigrated lymphocytes has remained constant comparedto day 2, in D (no additives) hardly any lymphocytes are detectable. Inthe partial culture C the number of lymphocytes has increased 20-foldcompared to A or B. The lymphocytes from C are isolated from the partialculture C and they are analysed on the basis of their surface markers.The population contains:

[0058] ca. 60% T lymphocytes (CD3+, CD4+, CD8+, CD19−)

[0059] ca. 25% NK cells (CD2+, CD3−, CD56+, CD19−) and

[0060] ca. 15% B lymphocytes (CD19+, CD3−).

LIST OF REFERENCE

[0061] Advances in Immunology 58 (1995) 87

[0062] Ayoub, I. A., Immunological Invest. 25 (1996) 129-151

[0063] Baughman-G., et al., Mol-Cell-Biol. 15 (1995) 4395-402

[0064] Callard, R. E., and Gearing, J. H., The Cytokine Facts Book,Academic Press, London 1994

[0065] Cerwenka, A. J. Immunol. 156 (1996) 459-464

[0066] EP-A 0 203 403

[0067] Immm. Rev. (1992) 82

[0068] Imm. Rev. (1993) 74

[0069] Inne, T. H., et al., J. Immunol. 148 (1992) 3847-3856

[0070] Jong de, R., Intern. Immunol. 6 (1994) 631-638

[0071] Kakalis, L. T., et al., FEBS-Lett. 362 (1995) 55-8

[0072] Kawamura, A., et al., J-Biol-Chem. 270 (1995) 15463-6

[0073] Kroemer, G., Advances in Immunology 58 (1995) 211-296

[0074] Kutnik, V., et al., Period. Biol. 92 (1990) 48

[0075] Pazderka-F., et al., Transpl-Immunol. (1996) 23-31

[0076] Pierson, B. A. et al., Blood 87 (1996) 180-189

[0077] Rusnak-F., et al., Bone-Marrow-Transplant. 17 (1996) 309-13

[0078] Su-Q., et al., Ren-Physiol-Biochem. 18 (1995) 128-39

[0079] Thiele and Lipsky, J. Immunol. 136 (1986) 1038-1048

[0080] WO90/10059

[0081] WO94/23014

[0082] Gonzales, R. G. et al., Biochemistry 19: 4299-4303 (1980)

1. Process for the production of lymphocytes, wherein lymphocytes arecultured, multiplied and isolated in a cell culture medium whichcontains a lymphocyte growth factor as well as additionallyaurintricarboxylic acid or a substance which binds to a cyclophilin andinhibits calcineurin in this complex.
 2. Process as claimed in claim 1,wherein the cyclophilin binding substance contains Cyclosporin®,Ascomycin® and/or Tacrolimus®.
 3. Process as claimed in claim 1 or 2,wherein T lymphocytes or NK lymphocytes are cultured and/or multipliedand the culture medium contains a T lymphocyte growth factor.
 4. Processfor the production of a mixture of T cells (CD2+, CD3+) and NK cells(CD2+, CD3−, CD16+), wherein mononuclear blood leucocytes are culturedin a culture medium which contains a T cell growth factor as well asadditionally aurintricarboxylic acid and/or a substance that binds tocyclophilin and inhibits calcineurin in this complex and the said Tcells and NK cells are isolated after a culture period of at least 10days.
 5. Process as claimed in claim 4, wherein Cyclosporin®, Ascomycin®or Tacrolimus® is used as the cyclophilin-binding substance.
 6. Processfor the production of a mixture of T cells (CD2+, CD3+) and NK cells(CD2+, CD3−, CD16+), wherein mononuclear blood leucocytes are culturedin a culture medium which contains a T cell growth factor and anapoptosis antagonist and the said T cells and NK cells are isolatedafter a culture period of at least 10 days.